Bottom Line:
Eleven mAbs inhibited BoNT/B LC proteolytic activity.The fine epitopes of selected mAbs were identified by alanine-scanning mutagenesis, revealing that inhibitory mAbs bound near the active site, substrate-binding site or the extended substrate-binding site.The results provide mAbs that could prove useful for intracellular reversal of paralysis and identify epitopes that could be targeted by small molecules inhibitors.

ABSTRACTExisting antibodies (Abs) used to treat botulism cannot enter the cytosol of neurons and bind to botulinum neurotoxin (BoNT) at its site of action, and thus cannot reverse paralysis. However, Abs targeting the proteolytic domain of the toxin could inhibit the proteolytic activity of the toxin intracellularly and potentially reverse intoxication, if they could be delivered intracellularly. As such, antibodies that neutralize toxin activity could serve as potent inhibitory cargos for therapeutic antitoxins against botulism. BoNT serotype B (BoNT/B) contains a zinc endopeptidase light chain (LC) domain that cleaves synaoptobrevin-2, a SNARE protein responsible for vesicle fusion and acetylcholine vesicle release. To generate monoclonal Abs (mAbs) that could reverse paralysis, we targeted the protease domain for Ab generation. Single-chain variable fragment (scFv) libraries from immunized mice or humans were displayed on yeast, and 19 unique BoNT/B LC-specific mAbs isolated by fluorescence-activated cell sorting (FACS). The equilibrium dissociation constants (KD) of these mAbs for BoNT/B LC ranged from 0.24 nM to 14.3 nM (mean KD 3.27 nM). Eleven mAbs inhibited BoNT/B LC proteolytic activity. The fine epitopes of selected mAbs were identified by alanine-scanning mutagenesis, revealing that inhibitory mAbs bound near the active site, substrate-binding site or the extended substrate-binding site. The results provide mAbs that could prove useful for intracellular reversal of paralysis and identify epitopes that could be targeted by small molecules inhibitors.

toxins-07-03405-f003: Fine epitopes of BoNT/B LC-binding mAbs. For each mAb, amino acids that result in loss of mAb binding are modeled on the crystal structure of BoNT/B LC (pdb ID: 1S0F). The BoNT/B LC is shown in cyan and the belt-binding groove in pink mesh. Structures B through F were generated by rotation of the BoNT/B LC strucutre in panel A as illustrated by the curved arrows. (A) The BoNT/B catalytic site is shown as an orange sphere. In each panel (A–F) the mAb name is colored to reflect the color of the corresponding residue in the epitope. Where a residue is shared by more than one mAb, the amino acid color is changed to reflect overlap (see legend, lower right). Panels (A,B) show the epitopes of mAbs in group 1 of epitope I; panel (C), mAbs in epitope cluster I, group 2; panel (D), mAbs in epitope cluster I, group 3; panel (E), mAbs in epitope cluster II; and panel (F), mAbs in epitope cluster III.

Mentions:
The fine epitopes of 17 of the mAbs were determined using yeast display [30], in order to define the binding sites on the BoNT/B LC that resulted in inhibition of endopeptidase activity. Mutations were randomly introduced into the BoNT/B LC gene using error prone PCR, and the resulting mutants were displayed on the surface of yeast. Each of the 19 mAbs was incubated separately with the yeast displayed BoNT/B LC library, and the yeast cells were sorted for loss of mAb binding. After three rounds of sorting with decreasing antigen concentration, individual yeast cells were analyzed to identify those not binding mAbs, and the BoNT/B LC gene was sequenced to identify the mutations responsible for loss of binding (Table 4). These mutations were then mapped onto the crystal structure of BoNT/B LC [31]. While a structure of substrate-bound BoNT/B LC has not been reported, one can use the BoNT/B belt as a substrate surrogate [32]. The belt-binding site is shown in pink in Figure 3. One of the mAbs in epitope cluster I, group 2 (34E8) could not be mapped due to limited expression of soluble scFv/IgG.

toxins-07-03405-f003: Fine epitopes of BoNT/B LC-binding mAbs. For each mAb, amino acids that result in loss of mAb binding are modeled on the crystal structure of BoNT/B LC (pdb ID: 1S0F). The BoNT/B LC is shown in cyan and the belt-binding groove in pink mesh. Structures B through F were generated by rotation of the BoNT/B LC strucutre in panel A as illustrated by the curved arrows. (A) The BoNT/B catalytic site is shown as an orange sphere. In each panel (A–F) the mAb name is colored to reflect the color of the corresponding residue in the epitope. Where a residue is shared by more than one mAb, the amino acid color is changed to reflect overlap (see legend, lower right). Panels (A,B) show the epitopes of mAbs in group 1 of epitope I; panel (C), mAbs in epitope cluster I, group 2; panel (D), mAbs in epitope cluster I, group 3; panel (E), mAbs in epitope cluster II; and panel (F), mAbs in epitope cluster III.

Mentions:
The fine epitopes of 17 of the mAbs were determined using yeast display [30], in order to define the binding sites on the BoNT/B LC that resulted in inhibition of endopeptidase activity. Mutations were randomly introduced into the BoNT/B LC gene using error prone PCR, and the resulting mutants were displayed on the surface of yeast. Each of the 19 mAbs was incubated separately with the yeast displayed BoNT/B LC library, and the yeast cells were sorted for loss of mAb binding. After three rounds of sorting with decreasing antigen concentration, individual yeast cells were analyzed to identify those not binding mAbs, and the BoNT/B LC gene was sequenced to identify the mutations responsible for loss of binding (Table 4). These mutations were then mapped onto the crystal structure of BoNT/B LC [31]. While a structure of substrate-bound BoNT/B LC has not been reported, one can use the BoNT/B belt as a substrate surrogate [32]. The belt-binding site is shown in pink in Figure 3. One of the mAbs in epitope cluster I, group 2 (34E8) could not be mapped due to limited expression of soluble scFv/IgG.

Bottom Line:
Eleven mAbs inhibited BoNT/B LC proteolytic activity.The fine epitopes of selected mAbs were identified by alanine-scanning mutagenesis, revealing that inhibitory mAbs bound near the active site, substrate-binding site or the extended substrate-binding site.The results provide mAbs that could prove useful for intracellular reversal of paralysis and identify epitopes that could be targeted by small molecules inhibitors.

ABSTRACTExisting antibodies (Abs) used to treat botulism cannot enter the cytosol of neurons and bind to botulinum neurotoxin (BoNT) at its site of action, and thus cannot reverse paralysis. However, Abs targeting the proteolytic domain of the toxin could inhibit the proteolytic activity of the toxin intracellularly and potentially reverse intoxication, if they could be delivered intracellularly. As such, antibodies that neutralize toxin activity could serve as potent inhibitory cargos for therapeutic antitoxins against botulism. BoNT serotype B (BoNT/B) contains a zinc endopeptidase light chain (LC) domain that cleaves synaoptobrevin-2, a SNARE protein responsible for vesicle fusion and acetylcholine vesicle release. To generate monoclonal Abs (mAbs) that could reverse paralysis, we targeted the protease domain for Ab generation. Single-chain variable fragment (scFv) libraries from immunized mice or humans were displayed on yeast, and 19 unique BoNT/B LC-specific mAbs isolated by fluorescence-activated cell sorting (FACS). The equilibrium dissociation constants (KD) of these mAbs for BoNT/B LC ranged from 0.24 nM to 14.3 nM (mean KD 3.27 nM). Eleven mAbs inhibited BoNT/B LC proteolytic activity. The fine epitopes of selected mAbs were identified by alanine-scanning mutagenesis, revealing that inhibitory mAbs bound near the active site, substrate-binding site or the extended substrate-binding site. The results provide mAbs that could prove useful for intracellular reversal of paralysis and identify epitopes that could be targeted by small molecules inhibitors.